This invention relates generally to an improved process for the production of L-amino acids (hereinafter "amino acids") from their .alpha.-keto acid precursors by biological transamination using aspartic acid as the amino donor. More specifically, the process disclosed herein is designed to improve the rate and yield of the transamination by increasing the rate of decomposition of oxalacetic acid, one of the reaction by-products. This results in a significant increase in the reaction rate such that substantially all of the precursor is consumed in the transamination. By eliminating the equilibrium constraint on the transamination and by allowing more precursor to be converted, amino acid yields of over 90 percent can be achieved. The reaction is catalyzed in this manner by the addition of metal ions, as solid metal particles or pieces comprising aluminum oxide, ferric oxide, lead oxide or zinc oxide, to the transamination reaction
system.
It is known that amino acid precursors may be converted enzymatically to their corresponding L-amino acids. For example, U.S. Pat. No. 3,183,170 (Kitai et al.) discloses the transamination of phenylpyruvic acid in the presence of a multi enzyme system obtained from various sources, including bacterial cells, dried cells, cell macerates or enzyme solutions. U.S. Ser. No. 520,632 (Fusee), filed Aug. 5, 1983, now abandoned, discloses the microbial transamination of .alpha.-keto acids to amino acids in fed-batch fermentations using conventional precursors to convert, for example, phenylpyruvic acid to L-phenylalanine, .alpha.-ketoisocaproic acid to L-leucine, .alpha.-ketoisovaleric acid to L-valine, etc.
The .alpha.-keto acid to amino acid biotransformation is an enzymatic transamination which results in the exchange of the amino group of the amino donor and the keto group of the precursor. It generally has been recognized that enzymatic transamination is an equilibrium reaction. For example, Oishi, in Ch. 16 of The Microbial Production of Amino Acids, (Yamada et al., Ed.), "Production from Precursor Keto Acids," pp. 440-46 (1972), notes that the use of aminotransferase has necessitated a high concentration of the amino donor for a high product yield. U.S. Pat. No. 3,183,170 (Kitai et al.) reports that in a transamination reaction using L-glutamic acid as the amino donor, the equilibrium is shifted favorably to the right by converting the alpha-keto glutaric acid resulting from the transamination back to L-glutamic acid by reductive amination as fast as the .alpha.-keto glutaric acid is formed.
In conventional transamination processes, a number of compounds have been used as the amino donor. Oishi, at pp. 435-52 of the Yamada et al. text, states that the best amino donors are L-aspartic acid, L-leucine, L-isoleucine and L-glutamic acid and that better results are obtained when these amino acids are used in combination than when they are used singly. U.S. Ser. No. 568,300 (Walter), filed on Jan. 5, 1984, now abandoned, discloses a process for driving a microbial transamination reaction towards completion by using a solution comprising approximately equimolar amounts of aspartic acid and phenylalanine precursor, pre-growing the microorganisms in the presence of the precursor, employing the biological catalyst in the form of dried cells, and/or increasing the biological catalyst loading of the system.
Oxalacetic acid (also known as oxaloacetic acid, oxosuccinic acid or keto succinic acid) is a by-product of enzymatic transamination when aspartic acid is used as the amino donor. Oxalacetic acid has been studied in other contexts and has been found to decompose by various mechanisms. Bessman, "Preparation and Assay of Oxalacetic Acid," Arch. Biochem., Vol. 26, pp. 418-21 (1950), reports spontaneous decomposition of oxalacetic acid, which is catalyzed by a number of substances, Krebs, "The Effect of Inorganic Salts on the Ketone Decomposition of Oxalacetic Acid," J. Biochem., Vol. 36, pp. 303-05 (1942), reports that various inorganic salts increase the rate of decomposition of oxalacetic acid into pyruvic acid and carbon dioxide.